In order to provide insight into how the oxidative DNA glycosylases may malfunction and thus lead to genome instability and cause cancer, we propose to use structural, biochemical and biological approaches to elucidate how these enzymes perform the first step In base excision repair. The long-term goal of this project is to not only provide fundamental insights into the mechanisms underpinning carcinogenesis but also to inform the human germ line SNP databases and allow us to predict with greater accuracy which SNP may lead to an increased risk of cancer. Moreover, understanding how variants In human tumors may affect function will inform prognosis and cancer treatment. The specific alms are as follows: Aim 1, A- To solve the crystal structures of the human members of the Fpg/Nei family NEIL1, NEIL2, and NEIL3, or close orthologs such as the mimivirus Nei proteins in complex with their DNA substrates. Towards this goal we have already obtained complexes of Mimivirus Neil with thymine glycol and spiroiminodihydantoin, which are the first examples of any Nei enzyme in complex with an oxidative DNA lesion. B- Once the structures are determined with substrate in the binding pocket, site directed mutants will be constructed for further biochemical analyses. C- To test the hypothesis that the NEILI, 2 and 3 variants Identified In Project 1 and Core A contribute to carcinogenesis by determining the biochemical characteristics of these variants such as substrate specificity and DNA binding properties and, where Indicated, their crystal structures. Aim 2. A- To solve the crystal structures of human members of the Nth superfamily or close orthologs in complex with their DNA substrates. Of note Is that no one has yet been able to crystallize hNTHI. B- To examine a select group of hNTHI germ line variants identified in Project 1 and Core A and through biochemical and structural analysis determine which variants may have potentially deleterious consequences. Structure/function studies from Project 2 will Inform the biological studies in Project 1 and provide insight into the interactions of glycosylases with nucleosomes In Project 4, Purified proteins and rapid enzyme analysis will be provided by Core B. Core A will help design and analyze enzyme kinetics experiments.